U.S. patent number 5,554,638 [Application Number 08/246,882] was granted by the patent office on 1996-09-10 for methods for improving therapeutic effectiveness of agents for the treatment of solid tumors and other disorders.
This patent grant is currently assigned to Apex Bioscience, Inc., Duke University, North Carolina State University. Invention is credited to Joseph Bonaventura, Joseph DeAngelo, Mark W. Dewhirst, Robert E. Meyer.
United States Patent |
5,554,638 |
Dewhirst , et al. |
September 10, 1996 |
Methods for improving therapeutic effectiveness of agents for the
treatment of solid tumors and other disorders
Abstract
The present invention is directed to the use of an inhibitor of
NO activity, such as a nitric oxide scavenger or an NO synthase
inhibitor, as an antitumor therapy to reduce tumor blood flow and
oxygenation. The invention is also directed to administration of a
nitric oxide scavenger or a nitric oxide synthase inhibitor to
enhance the effectiveness of tumor therapy with hypoxic or acidic
chemotherapeutic agents or hyperthermia. The invention is also
directed to the administration of a nitric oxide synthase substrate
to a subject previously administered a nitric oxide synthase
inhibitor, in order to selectively inhibit tumor perfusion. In a
specific example, administration of cell free hemoglobin, a nitric
oxide scavenger, in conjunction with mitomycin C, a hypoxic
cytotoxin, results in a significant delay in tumor growth of a
human tumor xenograft in a mouse compared to mitomycin C alone. In
another example, the administration of an inhibitor of nitric oxide
synthase followed by the administration of a substrate of the
enzyme causes a specific irreversible reduction of tumor blood
flow, while normal blood flow is restored.
Inventors: |
Dewhirst; Mark W. (Chapel Hill,
NC), Meyer; Robert E. (Cary, NC), Bonaventura; Joseph
(Beaufort, NC), DeAngelo; Joseph (Hamtramck, MI) |
Assignee: |
Duke University (Durham,
NC)
Apex Bioscience, Inc. (Durham, NC)
North Carolina State University (Raleigh, NC)
|
Family
ID: |
26747127 |
Appl.
No.: |
08/246,882 |
Filed: |
May 20, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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66756 |
May 24, 1993 |
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Current U.S.
Class: |
514/398; 514/560;
514/551; 514/456; 514/411; 514/565 |
Current CPC
Class: |
A61K
31/00 (20130101); A61P 35/00 (20180101); A61K
38/42 (20130101); A61K 31/195 (20130101); A61K
31/4045 (20130101); A61K 31/198 (20130101); A61K
41/0052 (20130101); A61P 43/00 (20180101); A61K
38/42 (20130101); A61K 31/4045 (20130101); A61K
31/4045 (20130101); A61K 31/198 (20130101); A61K
31/4045 (20130101); A61K 2300/00 (20130101); A61K
38/42 (20130101); A61K 2300/00 (20130101); Y10S
514/93 (20130101); Y10S 514/832 (20130101); Y10S
514/929 (20130101); Y10S 514/833 (20130101) |
Current International
Class: |
A61K
31/403 (20060101); A61K 38/42 (20060101); A61K
31/00 (20060101); A61K 38/41 (20060101); A61K
41/00 (20060101); A61K 31/185 (20060101); A61K
31/195 (20060101); A61K 31/198 (20060101); A61K
31/4045 (20060101); A61K 031/22 (); A61K
031/415 () |
Field of
Search: |
;514/565,560,551,398,411,456 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO88/03408 |
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May 1988 |
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WO |
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WO90/13645 |
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Nov 1990 |
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WO |
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WO93/08831 |
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May 1993 |
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WO |
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Primary Examiner: Ramsuer; Robert W.
Assistant Examiner: Peabody; John
Attorney, Agent or Firm: Pennie & Edmonds
Parent Case Text
This application is a continuation-in-part of U.S. patent
application Ser. No. 08/066,756, filed May 24, 1993, now pending.
Claims
What is claimed is:
1. A method for treating an animal having a vascularized solid
tumor comprising administering to an animal having a vascularized
solid tumor an amount of a nitric oxide synthase inhibitor
sufficient to reduce tumor blood flow and a therapeutically
effective amount of a hypoxic cytotoxin or acidotic cytotoxin,
wherein the animal is a mammal, the solid tumor is a sarcoma or
carcinoma, the nitric oxide synthase inhibitor inhibits or
suppresses nitric oxide synthesis by a nitric oxide synthase in
mammalian tissue, the hypoxic cytotoxin is more toxic to a
mammalian cell under hypoxic conditions than to the mammalian cell
not under hypoxic conditions, and the acidotic cytotoxin is more
toxic to the mammalian cell under acidotic conditions than to the
mammalian cell not under acidotic conditions.
2. The method according to claim 1 in which the nitric oxide
synthase inhibitor is administered at the same time as the
administration of the hypoxic cytotoxin or acidotic cytotoxin.
3. The method according to claim 1 in which the nitric oxide
synthase inhibitor is administered between 15 minutes and 60
minutes after the administration of the hypoxic cytotoxin or
acidotic cytotoxin.
4. The method according to claim 1, in which the nitric oxide
synthase inhibitor is an arginine analog; the hypoxic cytotoxin is
a mitomycin C, mitomycin C analog, or nitroimidazole; and the
acidotic cytotoxin is a cisplatin, cisplatin analog, bleomycin,
flavone acetic acid or etanidazole.
5. The method according to claim 4, in which the nitric oxide
synthase inhibitor is a tetrahydropterin analog, aminoguanidine,
methyl guanidine, N.sup.G -substituted arginine or N.sup.G,N.sup.G
-disubstituted arginine.
6. The method according to claim 5, in which the vascularized solid
tumor is selected from the group consisting of fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdosarcoma, colon
carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cellcarcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma; and the nitric oxide synthase inhibitor is selected
from the group consisting of N.sup.G -amino-L-arginine, N.sup.G
-nitro-L-arginine, N.sup.G -methyl-L-arginine, N.sup.G
-monomethyl-L-arginine, N.sup.G -ethyl-L-arginine, N.sup.G
-propyl-L-arginine, N.sup.G -butyl-L-arginine, N.sup.G
-nitro-L-arginine methyl ester, and N-iminoethyl-L-ornithine.
7. The method according to claim 2, which additionally comprises
administering a nitric oxide synthase substrate at the same time as
the administration of the nitric oxide synthase inhibitor.
8. The method according to claim 2, which additionally comprises
administering a nitric oxide synthase substrate after the
administration of the nitric oxide synthase inhibitor.
9. The method according to claim 3, which additionally comprises
administering a nitric oxide synthase substrate at the same time as
the administration of the nitric oxide synthase inhibitor.
10. The method according to claim 3, which additionally comprises
administering a nitric oxide synthase substrate after the
administration of the nitric oxide synthase inhibitor.
11. The method according to claim 7, 8, 9 or 10 in which the nitric
oxide synthase substrate is L-arginine.
12. The method according to claim 5 in which the nitric oxide
synthase inhibitor is a N.sup.G -substituted L-arginine or
N.sup.G,N.sup.G -disubstituted L-arginine.
13. The method according to claim 4 in which the nitric oxide
synthase inhibitor is administered in a dose in the range of about
0.1 mg/ml to about 100 mg/ml.
14. The method according to claim 12 in which the substituted
L-arginine is N.sup.G -monomethyl-L-arginine.
15. The method according to claim 4 which comprises administering a
therapeutically effective amount of the hypoxic cytotoxin mitomycin
C.
16. The method according to claim 4 in which the animal is a
human.
17. A pharmaceutical composition for treating an animal having a
vascularized solid tumor comprising an amount of a nitric oxide
synthase inhibitor sufficient to reduce tumor blood flow and a
therapeutically effective amount of a hypoxic cytotoxin or an
acidotic cytotoxin, wherein the animal is a mammal, the solid tumor
is a sarcoma or carcinoma, the nitric oxide synthase inhibitor
inhibits or suppresses nitric oxide synthesis by a nitric oxide
synthase in mammalian tissue, the hypoxic cytotoxin is more toxic
to a mammalian cell under hypoxic conditions than to the mammalian
cell not under hypoxic conditions, and the acidotic cytotoxin is
more toxic to the mammalian cell under acidotic conditions than to
the mammalian cell not under acidotic conditions.
18. The pharmaceutical composition of claim 17, in which the nitric
oxide synthase inhibitor is an arginine analog; the hypoxic
cytotoxin is a mitomycin C, mitomycin C analog, or nitroimidazole;
and the acidotic cytotoxin is a cisplatin, cisplatin analog,
bleomycin, flavone acetic acid or etanidazole.
19. The pharmaceutical composition of claim 18, in which the nitric
oxide synthase inhibitor is a tetrahydropterin analog,
aminoguanidine, methyl guanidine, N.sup.G -substituted arginine or
N.sup.G,N.sup.G -disubstituted arginine.
20. The pharmaceutical composition of claim 19, in which the
vascularized solid tumor is selected from the group consisting of
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
rhabdosarcoma, colon carcinoma, pancreatic cancer, breast cancer,
ovarian cancer, prostate cancer, squamous cell carcinoma, basal
cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma,
renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,
cervical cancer, testicular tumor, lung carcinoma, small cell lung
carcinoma, bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma, melanoma, neuroblastoma, and retinoblastoma; and the
nitric oxide synthetase inhibitor is selected from the group
consisting of N.sup.G -amino-L-arginine, N.sup.G -nitro-L-arginine,
N.sup.G -methyl-L-arginine, N.sup.G -monomethyl-L-arginine, N.sup.G
-ethyl-L-arginine, N.sup.G -propyl-L-arginine, N.sup.G
-butyl-L-arginine, N.sup.G -nitro-L-arginine methyl ester, and
N-iminoethyl-L-ornithine.
21. The pharmaceutical composition of claim 19 in which the nitric
oxide synthase inhibitor is a N.sup.G -substituted L-arginine or
N.sup.G,N.sup.G -disubstituted L-arginine.
22. The pharmaceutical composition of claim 21 in which the nitric
oxide synthase inhibitor is N.sup.G -monomethyl-L-arginine.
23. The pharmaceutical composition of claim 18 which comprises a
therapeutically effective amount of the hypoxic cytotoxin mitomycin
C.
24. The pharmaceutical composition of claim 18 further comprising a
pharmaceutically acceptable carrier.
Description
1. FIELD OF THE INVENTION
The present invention is directed to administration of a nitric
oxide (NO) scavenger or an NO synthesis inhibitor as an antitumor
therapy to reduce tumor blood flow and oxygenation or as an adjunct
therapy to enhance the effectiveness of tumor therapy with hypoxic
or acidic chemotherapeutic agents or hyperthermia. The invention is
also directed to the administration of an NO synthase substrate
subsequent to the administration of an NO synthase inhibitor, in
order to reverse the effect of the inhibitor on normal tissue.
2. BACKGROUND OF THE INVENTION
2.1. HYPOXIC AND HYPERTHERMIC TUMOR THERAPY
A relatively new and novel approach to solid tumor therapy has
involved the induction of tumor hypoxia following the
administration of drugs that are selectively cytotoxic to hypoxic
cells (Chaplin and Acker, 1987, Int. J. Rad. Oncology, Biol. Phys.
16:911-917; Brown and Koong, 1991, J. Natl. Cancer Inst.
83:178-185). The strategy typically involves the systemic
administration of a hypoxic cell cytotoxin, followed by the
administration of a drug that selectively reduces tumor blood flow.
The reduction in tumor blood flow traps the cytotoxic agent within
the tumor mass and increases its cytotoxicity via induction of
hypoxia (Babbs and DeWitt, 1981, Med. Instrum. 15:367-373; Chaplin
and Acker, 1987, Int. J. Rad. Oncology, Biol. Phys. 16:911-917;
Jain, 1988, Cancer Res. 48:2641-2658; Dewhirst et al., 1990, Int.
J. Hyperthermia 6:971-983).
Hyperthermia adjunct therapy for tumors is an area of active
investigation. An improvement in achievement of elevated
temperatures has been seen with reduction in tumor blood flow with
vasodilators (Dewhirst, et al., 1990, Int. J. Hyperthermia
6:971-983). Acidosis of tumors also leads to substantial
sensitization to heat killing (G. M. Hahn and E. C. Shiu, Int. J.
Radiat. Oncol. Biol Phys., 11:159-164, 1985).
Previous efforts to reduce tumor blood flow have focused primarily
on vasodilating agents such as hydralazine or nitroprusside. It has
been shown that reduction of systemic blood pressure leads to a
decrease in tumor blood flow while perfusion of normal tissues
either increases or is unaffected. The effects of these agents on
normal tissue perfusion is due to organ selectivity in the direct
effect of the drugs on arteriolar or venous tone as well as
systemic effects on cardiac output and arterial blood pressure. The
reduction in tumor perfusion is thought to be the result of
vascular collapse in tumors due to high interstitial fluid pressure
and high flow resistance in the presence of lowered arterial blood
pressure (Sevick and Jain, 1989, Cancer Res. 49:3506-3512). The
strategy has been shown to work effectively in murine systems and
in tumor bearing dogs (Dewhirst et al., 1990, Int. J. Hyperthermia
6:971-983; Prescott et al., 1990, Int. J. Hyperthermia 23:377-385),
but is directly related to the drop in blood pressure. However, the
blood pressure decrease required to observe reduced tumor
perfusion, to about 60% of normal blood pressure, makes the
approach relatively infeasible for clinical application. Such a
decrease in blood pressure is especially dangerous for elderly or
weak patients. The degree of reduction in systemic blood pressure
that is safe in patients is not enough to see an appreciable drop
in tumor blood flow.
2.2. NITRIC OXIDE (NO)
Nitric oxide (NO) is generally regarded as a radical, although the
chemical nature of NO remains an area of investigation. NO has
recently been identified as an endothelial relaxant factor. It
binds to guanylate cyclase in vascular smooth muscle and thereby
promotes vasodilation. Inhibition of NO synthase with N.sup.G
monomethyl L-arginine (L-NMA) or scavenging of NO with heme
proteins causes vasoconstriction and hypertension (Martin et al.,
1986, Br. J. Pharmacol. 89:563-571; Moncada et al., 1991, Pharm.
Rev. 43:109-142). Platelet aggregation is also decreased by NO
(Radomski et al., 1991, Cancer Res. 51:6073-6078). NO is involved
in neurotransmission in the central and peripheral nervous system
(Moncada et al., 1991, Pharmacol. Rev. 43:109-142.
Inhibitors of NO synthesis, such as N.sup.G -nitro-L-arginine and
N.sup.G -monomethyl-L-arginine have recently been shown to reduce
tumor blood flow (Andrade et al., 1992, Br. J. Pharmacol.
107:1092-1095; Wood et al, abstract presented at 41st Ann. Meeting
of Radiation Research Society, Dallas, Tex., Mar. 20-25, 1993; Wood
et al., abstract presented at 42nd Ann. Meeting of Radiation
Research Society, Nashville, Tenn., Apr. 29-May 5, 1994) this
effect could be prevented by prior injection of L-arginine (Andrade
et al., supra.), a precursor in the synthesis of NO. The NO
synthase inhibitor L-NMA was found to increase tumor resistance to
X-rays (Wood et al., supra).
Citation or identification of any reference herein shall not be
construed as an admission that such reference is available as prior
art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention is directed to a method for treating a
subject having a solid tumor comprising administering to the
subject an amount of an inhibitor of vascular nitric oxide
activity, such as a nitric oxide scavenger or a nitric oxide
synthase inhibitor, sufficient to decrease tumor blood flow or
oxygenation. In another aspect, the invention is directed to a
method for treating a subject having a solid tumor comprising
administering (a) an amount of an inhibitor of vascular nitric
oxide activity sufficient to decrease tumor blood flow or
oxygenation, and (b) a hypoxic or acidotic chemotherapeutic
agent.
In another aspect, the invention is directed to a method for
treating a subject having a solid tumor comprising administering to
the subject an amount of an inhibitor of vascular nitric oxide,
such as a nitric oxide scavenger or a nitric oxide synthase
inhibitor, in an amount sufficient to decrease tumor blood flow or
tumor oxygenation, and administering hyperthermia therapy.
In another aspect, the invention is directed to a method for
treating a subject having a solid tumor comprising administering to
the subject an amount of a competitive inhibitor of nitric oxide
synthase (e.g., a substrate analog) in an amount sufficient to
decrease tumor blood flow or tumor oxygenation, and, subsequently,
administering a substrate of nitric oxide synthase, in order to
selectively decrease the reduction of blood flow and oxygenation in
normal tissue. In a preferred aspect, the substrate is administered
in an amount effective to restore normal blood flow and oxygenation
to normal tissue.
In a further aspect, the present invention provides a
pharmaceutical composition for treating solid tumors comprising a
nitric oxide scavenger or a nitric oxide synthase inhibitor and a
hypoxic cytotoxin or acidotic cytotoxin.
In a further aspect, the present invention provides a
pharmaceutical composition for treating solid tumors comprising a
substrate of nitric oxide synthase. The invention also provides a
kit comprising in separate containers: an inhibitor of vascular
nitric oxide synthase in a pharmaceutically acceptable form, and a
substrate of nitric oxide synthase in a pharmaceutically acceptable
form.
The present invention combines therapeutic modalities to
selectively target various solid tumor populations that vary in
their microenvironmental conditions. One advantage of the present
invention is that the tumor-specific toxicity of hypoxic cytotoxins
toward hypoxic tumors can be enhanced, thus increasing the
efficiency of the hypoxic cytotoxin. Increased toxic efficiency can
allow lower doses, and thus, reductions in toxic side effects, or
more effective therapy leading to better outcomes. Another
particular advantage of the present invention is that it permits
therapy of aerobic cells using hypoxic cytotoxins. Furthermore, the
invention allows for powerful combination therapies, for example
radiation, which is effective against aerobic tumor cells, with
hyperthermia and drugs that are effective against hypoxic and
acidotic cells. This combination achieves a more uniform cell kill
over all physiologic subtypes than any of the treatments alone.
Another advantage of the invention is that it allows applications
of treatment regimens against subpopulations of tumor cells while
having minimum effects on normal tissue.
The present invention is illustrated by way of example by the
demonstration of the feasibility of the therapeutic approach of
combining NO inhibition with the hypoxic cell cytotoxin mitomycin
C. In a specific example, infra, a trend toward enhancement of
tumor growth delay was observed when tumor-bearing animals were
treated with stroma-free hemoglobin (a NO scavenger) 40 min after
administration of mitomycin C, as compared with mitomycin C alone.
In another specific example, infra, NO synthase inhibition by L-NMA
reduced blood flow in tumor tissue while the effect of the
inhibitor was selectively reversed in normal tissue by the
administration of L-arginine.
4. DESCRIPTION OF THE FIGURES
FIG. 1. Effect of intravenous administration of L-NMA on mean
arteriolar blood pressure (MAP) (open squares, open circles, and
crossed-squares) and heart rate (open diamonds, open triangles and
closed diamonds) in Fischer 344 rats. Data are from 3
experiments.
FIG. 2. Effect of intravenous cell free hemoglobin solution on
arterial blood pressure and heart rate in Fischer 344 rats. Data
were pooled from 5 experiments, and include measurements of
diastolic blood pressure (open squares), systolic blood pressure
(open diamonds) and mean blood pressure (open circles) in mm Hg,
and heart rate (open triangles) in beats per minute.
FIG. 3. Average heart rate (beats/min) over time of rats treated
with albumin (open squares), hemoglobin P.sub.50 9 (solid diamonds)
and hemoglobin P.sub.50 32 (solid squares). Data are averaged from
three rats per experimental group.
FIG. 4. Mean arteriolar pressure (MAP) in mm Hg over time for rats
treated with albumin (open squares), hemoglobin p50 9 (solid
diamonds) and hemoglobin p50 32 (solid squares). Data are averaged
from three rats per experimental group.
FIG. 5. Average hematocrit of rats treated with albumin (open
squares), hemoglobin p50 9 (solid diamonds) and hemoglobin p50 32
(solid squares). Data are averaged from three rats per experimental
group.
FIG. 6. Muscle oxygenation changes (mm Hg) in rats treated with
albumin (open squares), hemoglobin p50 9 (solid diamonds) and
hemoglobin p50 32 (solid squares). Data are averaged from three
rats per experimental group.
FIG. 7. Average tumor oxygenation changes (mm Hg) in three rats
treated with albumin.
FIG. 8. Average tumor oxygenation changes (mm Hg) in three rats
treated by hemoglobin p50 9.
FIG. 9. Average tumor oxygenation changes (mm Hg) in three rats
treated with hemoglobin p50 32.
FIG. 10: Relative change in microvessel diameter after 60 min
superfusion of L-NMA followed by 60 min superfusion of L-arginine.
L-NMA significantly reduced diameters for all types of tumor
preparation venules (tumor center, tumor periphery, normal near
tumor) as well as for venules in control preparations (all
p<0.05). Superfusion of L-arginine had negligible restoring
effect on tumor preparation venules, but returned control venules
to baseline diameter. Symbols represent mean.+-.SEM. Open squares:
tumor center; open diamond: tumor periphery; open circle: normal,
near tumor; open triangle: control, no tumor.
FIG. 11: Relative change in microvessel RBC velocity after 60 min
superfusion of L-NMA followed by 60 min superfusion of L-arginine.
L-NMA reduced RBC velocity of control and tumor center venules from
baseline (both p<0.05). L-arginine returned RBC velocity to
baseline levels in tumor periphery vessels, normal vessels near
tumors, and control vessels, but RBC velocity in tumor center
venules remained significantly reduced from baseline (p<0.05).
Symbols represent mean.+-.SEM. Open squares: tumor center; open
diamond: tumor periphery; open circle: normal, near tumor; open
triangle: control, no tumor.
FIG. 12: Relative change in microvessel flow after 60 min
superfusion of L-NMA followed by 60 min superfusion of L-arginine.
L-NMA reduced relative flow 43% in tumor center and peripheral
tumor vessels, and 83% in control vessels. L-arginine restored
peripheral tumor flow to the same levels observed in normal vessels
near tumors. Flow in central tumor vessels continued to decrease in
the presence of L-arginine. The graph is provided to illustrate the
interaction between diameter and RBC velocity. Open squares: tumor
center; open diamond: tumor periphery; open circle: normal, near
tumor; open triangle: control, no tumor.
FIG. 13. Relative change in vessel length density after 60 min
superfusion of L-NMA followed by 60 min superfusion of L-arginine.
Although not statistically significant, L-NMA may reduce vessel
length density in tumor vessels (p=0.07, tumor center; p=0.08 for
both tumor periphery and normal vessels near tumors). Vessel length
density further decreased following L-arginine for both tumor
center and peripheral tumor vessels (p=0.01 and p=0.05,
respectively). Symbols represent mean.+-.SEM. Open squares: tumor
center; open diamond: tumor periphery; open circle: normal, near
tumor; open triangle: control, no tumor.
FIG. 14: Percent of vessels showing intermittent flow or stasis
after 60 min superfusion of L-NMA followed by 60 min superfusion of
L-arginine. L-NMA increased intermittent flow and stasis in central
tumor vessels relative to baseline (p=0.03). Intermittent flow and
stasis may increase with L-NMA in peripheral tumor vessels and
normal vessels near tumors (p=0.06 for both). L-arginine returned
intermittent flow and stasis to baseline levels for all vessel
types. Symbols represent mean.+-.SEM. Open squares: tumor center;
open diamond: tumor periphery; open circle: normal, near tumor;
open triangle: control, no tumor.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to the use of an inhibitor of
nitric oxide (NO) activity, such as a NO scavenger or an NO
synthase inhibitor, as an antitumor therapy to reduce tumor blood
flow and oxygenation, or as an adjunct therapy in the treatment of
solid tumors with (1) a chemotherapeutic agent, in particular a
hypoxic cytotoxin or an acidotic cytotoxin, and/or with (2)
hyperthermia.
According to the present invention, inhibition of NO, either with
inhibitors of NO synthesis or NO scavengers, selectively reduces
blood flow in solid tumors, and leads to irreversible vascular
stasis in some tumor vessels. The effects of NO reduction in normal
tissue are less frequent and much less extensive, indicating that
reduction in NO concentration creates a selective effect in the
tumor. Inhibitors of NO synthesis and NO scavengers cause minimal
side effects, and these side effects consist primarily of a mild
and transient pressor effect.
The present inventors have discovered that NO synthase inhibition
can be used to effect preferential tumor blood flow reduction,
since the tumor blood flow reduction is irreversible even upon
administration of an NO synthase substrate, while blood flow
reduction in normal tissue is reversed by administration of such a
substrate. The present invention provides a method selectively to
decrease blood flow reduction in normal tissue resulting from the
administration of a nitric oxide synthase inhibitor (optionally,
the inhibitor being administered concurrently or sequentially with
the administration of a hypoxic or acidotic chemotherapeutic agent
or with hyperthermia) by administering a substrate of NO
synthase.
According to the invention, therapeutic administration of an NO
scavenger or NO synthase inhibitor in conjunction with
administration of a chemotherapeutic agent leads to enhanced
anti-tumor chemotherapeutic effectiveness. Therapeutic
administration of an NO scavenger or NO synthase inhibitor in
conjunction with hyperthermia therapy leads to enhanced toxicity
toward the solid tumor. The hypoxic cytotoxins are cytotoxins that
are effectively cytotoxic to hypoxic or acidic cells or under
hypoxic conditions. The acidotic cytotoxins are agents whose
cytotoxicity is enhanced under acid pH conditions.
The present inventors have discovered that the physiological
consequences of tumor blood flow reduction include reduction in the
heat transfer capacity of a tumor, and the induction of hypoxia and
acidosis. Hypoxia and acidosis contribute to hyperthermia
cytotoxicity. Hyperthermia cytotoxicity is greatly enhanced in
cells that demonstrate acute drops in pH, even when the magnitude
of the drop is only a few tenths of pH.
According to the present invention, inhibition of NO, either with
antagonists of NO synthase or scavenging of NO (e.g., with stroma
free hemoglobin) reduces tumor blood flow and leads to tumor
vascular stasis. The effect is achieved in tumor with only a mild
and transient systemic pressor effect. Although not intending to be
bound by any particular theory, it is believed that two mechanisms
are responsible for this effect: (1) reduction in NO causes
vasoconstriction in normal arterioles that feed the tumor, and (2)
platelet aggregation is stimulated preferentially within tumor
vessels, leading to microthrombus formation.
5.1. NO SCAVENGERS
The present invention contemplates the use of any NO scavenger as
an antitumor therapy to reduce tumor blood flow and oxygenation or
as an adjunct therapy to potentiate or enhance the chemotherapeutic
effect of a hypoxic or acidic cytotoxin, or to enhance the effect
of hyperthermia therapy. As used herein, the term "NO scavenger"
refers to any molecular entity that binds with free NO so as to
reduce the concentration of NO locally or systemically. Such
scavengers include, but are not limited to, metalloproteins, in
particular heme containing proteins such as but not limited to
hemoglobin, myoglobin, cytochrome-P-450, heme albumin,
heme-containing peptides such as undecapeptide of cytochrome C, as
well as water soluble hemoglobin analogs such as strapped heme
(e.g., Traylor and Traylor, 1982, Ann. Rev. Biophys. Bioeng.
11:105-127) and picket fence porphyrin (Collman et al., 1975 J. Am.
Chem. Soc. 97:1427-1439). In a preferred embodiment, the scavenger
selected for use is one which, in vivo, in vitro, or animal model
experiments, is shown to be capable of causing vascular stasis that
is not reversed by L-arginine.
Use of many NO scavengers according to the present invention has
the advantage of restricting NO reduction to the vasculature,
without affecting intracellular NO production or extravascular NO
activity. Thus, NO activity as a transduction mechanism for soluble
guanylate cyclase in the nervous system and the function of immune
cells, such as macrophages, will be minimally affected, thus
reducing possible side effects of therapy with an NO scavenger.
Macromolecular NO scavengers such as hemoglobin can become trapped
in the perivascular space of a solid tumor, since the tumor
vasculature is very leaky compared to normal tissue. Macromolecules
such as hemoglobin do not readily pass into the perivascular space
of normal tissue, so they are less likely to be trapped there.
Trapping of an NO scavenger, such as hemoglobin, in the
perivascular space of the tumor tends to restrict the NO scavenging
effect to the tumor, thus enhancing tumor hypoxia with minimal
effects on normal tissue.
In a preferred aspect of the invention, the NO scavenger is cell
free hemoglobin (CFHb), also referred to as stroma free hemoglobin.
Stroma-free hemoglobin may be obtained using procedures known in
the art (see for example, PCT Application Publication No. WO
88/03408, published May 19, 1988; U.S. Pat. No. 4,001,401; Feola et
al., 1983, Surgery Gynecology and Obstetrics 157:399-408; De Venuto
et al., 1979, Surgery Gynecology and Obstetrics 149:417-436). For
example, stroma-free hemoglobin may be obtained as follows: (a)
obtaining whole blood; (b) separating red blood cells from other
components of whole blood; (c) isolating the hemoglobin from the
erythrocytes; and (d) separating the hemoglobin from stroma and
other impurities.
Stroma-free hemoglobin can be prepared starting with erythrocytes
in freshly drawn, outdated, or frozen packed cells or whole blood.
The blood should be drawn in a sterile fashion into containers with
sufficient anticoagulant activity to prevent clot formation.
In one embodiment, the erythrocytes are washed in a saline solution
and centrifuged to separate red blood cells from white blood cells
and to additionally remove free proteins (Feola et al., 1983,
Surgery Gynecology and Obstetrics 157:399-408). In another
embodiment, the red cells may be separated from other erythrocytes
by passing through a semi-continuous type centrifuge as described
in PCT Application Publication No. WO 88/03408, published May 19,
1988.
Hemoglobin may be isolated in one embodiment by diluting the red
blood cell solution in water or an organic solvent at about
2.degree. to about 10.degree. C. to separate the hemoglobin in red
blood cells from all cell debris (PCT Application Publication No.
WO 88/03408, published May 19, 1988; U.S. Pat. No. 4,001,401; Feola
et al., 1983, Surgery Gynecology and Obstetrics 157:399-408). In
another embodiment, the hemoglobin is precipitated as a zinc
complex by the addition of a zinc salt to a hemoglobin solution (De
Venuto et al., 1979, Surgery Gynecology and Obstetrics.
149:417-436).
The isolated hemoglobin may in one embodiment be purified by
ultrafiltration through for example a 0.5.mu. pore filter which
retains the cellular components and passes the hemoglobin.
Hemoglobin may also be obtained through other procedures known in
the art. For example, bacterial strains (see for example Nagai and
Hoffman, U.S. Pat. No. 5,028,588, issued Jul. 2, 1991) or yeast
(see for example PCT Application Publication No. WO90/13645,
published Nov. 15, 1990; U.S. patent application Ser. No.
07/876,290, filed Apr. 29, 1992, entitled "Expression of
Recombinant Hemoglobin or Hemoglobin Variants in Yeast"), or other
eukaryotic organisms may be engineered to produce hemoglobin by
recombinant DNA techniques.
The hemoglobin may be, for example, any human hemoglobin or
hemoglobin variant, including but not limited to HbA (alpha.sub.2
beta.sub.2), HbA2 (alpha.sub.2 delta.sub.2), HbF (alpha.sub.2
gamma.sub.2), Hb Barts (gamma.sub.4), HbH (beta.sub.4), and Hb
Portland I (zeta.sub.2 gamma.sub.2), Hb Portland II (zeta.sub.2
beta.sub.2), Hb Portland III (zeta.sub.2 delta.sub.2), Hb Gower I
(zeta.sub.2 epsilon.sub.2), and Hb Gower II (alpha.sub.2
epsilon.sub.2); as well as any other animal hemoglobin, e.g.,
bovine or porcine hemoglobin. Hemoglobin dimers are also useful,
but unmodified hemoglobin monomers or dimers can lead to
unacceptable renal toxicity.
The hemoglobin used in the method of the present invention may be
chemically modified using procedures known in the art to form
polymers of Hb tetramers (to increase half-life in circulation,
e.g., Hb Porto Alegre), or to increase tetramer stability (to
decrease renal toxicity) and/or lower oxygen affinity. Examples of
chemical modifications to increase the tetramer stability include
but are not limited to crosslinking with polyalkylene glycol
(Iwashita, U.S. Pat. Nos. 4,412,989 and 4,301,144), with
polyalkylene oxide (Iwasake, U.S. Pat. No. 4,670,417); with a
polysaccharide (Nicolau, U.S. Pat. Nos. 4,321,259 and 4,473,563);
with inositol phosphate (Wong, U.S. Pat. Nos. 4,710,488 and
4,650,786); with a bifunctional crosslinking agent (Morris et al.,
U.S. Pat. No. 4,061,736); with.insulin (Ajisaka, U.S. Pat. No.
4,377,512); and with a crosslinking agent so that the hemoglobin
composition is intramolecularly crosslinked between lys 99
alpha.sub.1, and lys 99 alpha.sub.2 (Walder, U.S. Pat. No.
4,598,064). Examples of chemical modifications to decrease the
oxygen affinity of isolated hemoglobin include but are not limited
to polymerization with pyridoxal phosphate (Sehgal et al., 1984,
Surgery 95:433-438) and using reagents that mimic
2,3-diphosphoglycerate (DPG) (Bucci et al., U.S. Pat. No.
4,584,130).
In a further embodiment, the hemoglobin used in the method of the
present invention may be a hemoglobin variant, a hemoglobin
comprising a globin chain whose nucleotide sequence has been
altered in such a fashion so as to result in the alteration of the
structure or function of the hemoglobin, but so that the hemoglobin
still remains functionally active as defined by the ability to
reversibly bind to nitric oxide. Categories of hemoglobin variants
include but are not limited to variants which autopolymerize;
variants in which the tetramer does not dissociate under
physiological conditions in vivo (e.g., Hb Rainier, beta-145
tyrosine is replaced by cysteine); variants with lowered intrinsic
oxygen affinity, i.e., a hemoglobin having a p50 (p50 is the
partial pressure of oxygen which results in 50% saturation of
oxygen binding in hemoglobin) of at least about 10 mm Hg under
physiological conditions (e.g., Hb Chico, beta-66 lysine is
replaced by threonine; Hb Raleigh, beta-1 valine is replaced by
alanine; Hb Titusville, alpha-94 aspartate is replaced by
asparagine; Hb Beth Israel, beta-102 asparagine is replaced by
serine; and Hb Kansas, beta-102 asparagine is replaced by
threonine); variants that are stable in alkali (e.g.,
Motown/Hacettepe beta-127 or glutamine is replaced by glutamic
acid); variants that are stable in acid; variants which have a
lowered binding affinity to haptoglobin; variants with an increased
intrinsic oxygen affinity, i.e., a hemoglobin having a P50 of at
most about 1 mm Hg under physiological conditions (e.g., HbA Deer
Lodge, beta-2 histidine is replaced by arginine, Labossiere et al.,
1972, Clin. Biochem. 5:46-50; HbA Abruzzo, beta-143 histidine is
replaced by arginine, Tentori et al., 1972, Clin. Chim. Acta
38:258-262; and HbA McKees Rocks, the coding sequence is altered so
that the sequence encoding beta-145 tyrosine is replaced by a
termination codon (Winslow et al., 1976, J. Clin. Invest.
57:772-781).
Acid stable hemoglobin variants may include those that replace the
histidine at the alpha-103 position with an amino acid that is not
ionized in acid (Perutz, 1974, Nature 247:341). Examples of such
amino acids include serine, threonine, leucine, and alanine.
Haptoglobin nonbinding variants are those with variation in the
alpha-Hb sequence in the region of amino acid numbers 121-127. This
sequence has been shown to be involved in the binding of
haptoglobin (McCormick and Atorssi, 1990, J. Prot. Chem.
9:735).
The globin variants may be produced by various methods known in the
art. The manipulations which result in their production can occur
at the gene or protein level. The globin may be altered at the gene
level by site-specific mutagenesis using procedures known in the
art. One approach which may be taken involves the use of synthetic
oligonucleotides to construct variant globins with base
substitutions. In one embodiment, a short oligonucleotide
containing the mutation is synthesized and annealed to the
single-stranded form of the wild-type globin gene (Zoller and
Smith, 1984, DNA 3:479-488). The resulting short heteroduplex can
serve as primer for second strand synthesis by DNA polymerase. At
the 5' end, a single stranded nick is formed which is closed by DNA
ligase. In another embodiment, two complementary oligonucleotides
are synthesized, each containing the mutant sequence. The duplex
that forms after annealing these complementary oligonucleotides can
be joined to a larger DNA molecule by DNA ligase provided that the
ends of both molecules have complementary single-stranded "sticky"
ends. Another approach which may be taken involves introducing a
small single-stranded gap in the DNA molecule followed by
mis-repair DNA synthesis, i.e., the misincorporation of a
non-complementary nucleotide in the gap (Botstein and Shortle,
1985, Science 229:1193). The incorporation of a thiol nucleotide
into the gap may minimize the excision of the noncomplementary
nucleotide. Alternatively, a globin variant may be prepared by
chemically synthesizing the DNA encoding the globin variant using
procedures known in the art (see for example Froehler, 1986, Nucl.
Acids Res. 14:5399-5407 and Caruthers et al., 1982, Genetic
Engineering, J. K. Setlow and A. Hollaender eds., Plenum Press, New
York, vol. 4, pp. 1-17). In a preferred embodiment, fragments of
the variant globin are chemically synthesized and these fragments
are subsequently ligated together. The resulting variant globin
strands may be amplified using procedures known in the art, e.g.,
PCR technology, and subsequently inserted into a cloning vector as
described supra. In a specific embodiment, site-specific mutants
may be created by introducing mismatches into the oligonucleotides
used to prime the PCR amplification (Jones and Howard, 1990,
Biotechniques 8:178-180).
Manipulations of the globin sequence may be carried out at the
protein level. Any of numerous chemical modifications may be
carried out by known techniques including but not limited to
specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin, papain, V8 protease, NaBH.sub.4 ; acetylation,
formylation, oxidation, reduction; etc. Alternatively, the variant
globin protein may be chemically synthesized using procedures known
in the art, such as commercially available peptide synthesizers and
the like. Such standard techniques of polypeptide synthesis can be
found described in such publications as Merrifield, 1963, J. Chem.
Soc. 85:2149-2154 and Hunkapillar et al., 1984, Nature (London)
310:105-111.
Any of the foregoing variants can be tested for NO scavenging
activity, e.g., according to the assay described in Sections 6 and
7, infra.
The hemoglobin that is used is preferably of mammalian origin, and
can be from pigs, cows, dogs, cats, mice, rats, horses, primates
such as monkeys and chimpanzees, and is most preferably human. In a
specific embodiment, infra, the NO scavenger that is used is cell
free hemoglobin of bovine origin.
5.2. INHIBITORS OF NO SYNTHESIS
The present invention contemplates the use of any inhibitor of NO
synthase as an antitumor therapy to reduce tumor blood flow and
oxygenation or as an adjunct therapy to potentiate or enhance the
chemotherapeutic effect of a hypoxic or acidic cytotoxin, or to
enhance the effect of hyperthermia therapy. As used herein, the
term "NO synthase inhibitor" refers to any competitive or
noncompetitive inhibitor of NO synthase.
In a preferred aspect of the invention, the NO synthase inhibitor
is an arginine analog, such as aminoguanidine or methyl guanidine,
and N.sup.G -substituted arginine or an N.sup.G,N.sup.G
-disubstituted arginine. Preferably, the substituted arginine is of
the L configuration. Examples of substituted L-arginines for use as
NO synthase inhibitors according to the invention include, but are
not limited to, N.sup.G -amino-L-arginine, N.sup.G
-nitro-L-arginine, N.sup.G -alkyl-L-arginines such as N.sup.G
-methyl-L-arginine or N.sup.G -monomethyl-L-arginine (often
abbreviated NMMA, L-NMA or L-NMMA), N.sup.G -ethyl-L-arginine,
N.sup.G -propyl-L-arginine, or N.sup.G -butyl-L-arginine, N.sup.G
-nitro-L-arginine methyl ester (often abbreviated NAME or L-NAME),
and N-iminoethyl-L-ornithine (often abbreviated NIO or L-NIO).
These inhibitors are available from commercial sources, e.g.,
Calbiochem, Sigma, and Aldrich.
In another embodiment, an inhibitor of the NO synthase cofactor
tetrahydropterin can be used. One such inhibitor is
aminopterin.
In a specific example, infra, the NO synthase inhibitor that is
used is N.sup.G -monomethyl-L-arginine, abbreviated L-NMA).
5.2.1. OTHER INHIBITORS OF NO ACTIVITY
In addition to inhibitors of NO synthase, the present invention
contemplates use of inhibitors of the second messenger system
activated by NO, particularly the second messengers (downstream
signal mediators) guanylate cyclase and cyclic GMP. A nonlimiting
example of guanylate cyclase inhibition is methylene blue. Cyclic
GMP activity can be inhibited by aminoguanidine, such as M&B
22948.
5.2.2. REVERSAL OF NO SYNTHESIS INHIBITION IN NORMAL TISSUE
The present invention also contemplates the therapeutic
administration of an NO synthase inhibitor (a competitive
inhibitor, e.g., a substrate analog) followed by administration of
an NO synthase substrate so as to selectively reverse any effect of
the inhibitor on normal tissue. As shown by way of example infra,
the effect of the inhibitor on the tumor tissue is
irreversible.
No synthase substrates which can be used include but are not
limited to guanidino succinate and L-arginine. In a specific
embodiment, the NO synthase substrate that is used is
L-arginine.
5.3. THERAPEUTIC METHODS AND COMPOSITIONS
The present invention is directed to methods for treating a subject
having a solid tumor comprising administering an inhibitor of NO
activity as an antitumor therapy to reduce tumor blood flow and
oxygenation or as an adjunct therapy to potentiate or enhance the
chemotherapeutic effect of a hypoxic or acidic cytotoxin, or to
enhance the effect of hyperthermia therapy. The invention is also
directed to the administration of a NO synthase inhibitor (a
competitive inhibitor, e.g., a substrate analog) followed by the
administration of a NO synthase substrate so as to alleviate any
effects of the inhibitor on normal tissue. Preferably the subject
is an animal, more preferably a mammal, and most preferably a
human. However, the present invention is also directed to treatment
of tumors of domestic animals, such as feline or canine subject,
and farm animals, such as but not limited to bovine, equine and
porcine subjects. In a specific embodiment, infra, the therapeutic
method of the invention is effective to inhibit growth of a human
tumor xenograft in a mouse.
Preferably the therapeutic methods of the invention result in an
increase in tumor regression rate (response rate), local tumor
control and/or reduction in the frequency of or elimination of
growth of metastases. The therapeutic approach is directed to
tumors that are large enough to be vascularized. Enhanced local
control of a vascularized tumor reduces the probability of
metastases by causing irreversible vascular stasis and by enhanced
tumor cell kill. The therapeutic approach could be used for small
metastases as well, in conjunction with systemic chemotherapy of
the types discussed above.
According to the present invention, the NO scavenger, as described
in Section 5.1, supra, or the NO synthase inhibitor, or the NO
synthase substrate can be administered parenterally, i.e., via an
intraperitoneal, intravenous, perioral, subcutaneous,
intramuscular, intraarterial, etc. The preferred route of
parenteral administration is intravenous. Preferably the NO
scavenger or the NO synthase inhibitor or the NO synthase substrate
is prepared in an admixture with a pharmaceutically acceptable
carrier. The term "carrier" refers to diluents, excipients and the
like for use in preparing admixtures of a pharmaceutical
composition. Pharmaceutically acceptable carriers include but are
not limited to sterile water, saline, buffered saline, dextrose
solution, preferably such physiologically compatible buffers as
Hank's or Ringer's solution, physiological saline, a mixture
consisting of saline and glucose, and heparinized
sodium-citrate-citric acid-dextrose solution and the like. As used
herein, the term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans.
Techniques and formulations for administering the compositions may
be found in Remington's Pharmaceutical Sciences, Meade Publishing
Col., Easton, Pa., latest edition.
Generally, the NO scavenger or NO synthase inhibitor or NO synthase
substrate is administered in a single bolus dose whether
administered alone or in connection with chemotherapy or
hyperthermia therapy, although the present invention also
contemplates sustained administration, e.g., via an IV drip or
pump, or administration in multiple boluses.
In a specific aspect, the invention also provides kits comprising
in a container the NO scavenger or NO synthase inhibitor or NO
synthase substrate in pharmaceutically acceptable form; e.g.,
lyophilized or in admixture with a pharmaceutically acceptable
carrier. In one embodiment, a kit comprises in separate containers
effective amounts of an NO synthase inhibitor and an NO synthase
substrate.
5.3.1. ADMINISTRATION OF INHIBITORS OF NO ACTIVITY
The present invention contemplates administration of an inhibitor
of NO activity, such as a nitric oxide scavenger or an NO synthase
inhibitor, as an antitumor therapy to reduce tumor blood flow and
oxygenation.
Preferably the therapeutic methods of the invention result in an
increase in tumor regression rate (response rate), local tumor
control and/or reduction in the frequency of or elimination of
growth of metastases. The therapeutic approach is directed to
tumors that are large enough to be vascularized. Enhanced local
control of a vascularized tumor reduces the probability of
metastases by causing irreversible vascular stasis, and by enhanced
tumor cell kill.
The dose of the NO scavenger or NO synthase inhibitor to be
administered is a dose effective to reduce blood flow or the level
of oxygenation in a tumor, e.g., as detected by such ability in
vivo, or as extrapolated from in vitro assays or from the animal
model systems as described in Sections 6 and 7, infra. For example,
if the NO scavenger is cell free hemoglobin, in one embodiment, the
dose (in g of scavenger per mass of the subject in kg) can be from
about 0.01 g/kg to about 10 g/kg; in a specific example, infra, the
dose is about 0.1 g/kg, which represents less than 5% of the total
blood volume of the animal. If the NO synthase inhibitor is a
substituted arginine, in a specific embodiment, the dose can be
from about 0.1 mg/kg to about 100 mg/kg. In a specific embodiment,
infra, the synthase inhibitor is L-NMA administered at a dose of
3.0 mg/kg.
The present invention also contemplates the administration of an NO
synthase inhibitor, followed by the administration of a NO synthase
substrate, so as to effect the selective reduction of tumor blood
flow and increased tumor hypoxic cell fraction, while tending to
restore normal tissue blood flow. Preferably, the NO synthase
substrate is administered from 10 minutes to 4 hours after the
administration of the NO synthase inhibitor.
5.3.2. ADMINISTRATION WITH HYPOXIC AND ACIDIC CYTOTOXINS
The present invention contemplates administration of an NO
scavenger or an NO synthase inhibitor in conjunction with a hypoxic
or acidic chemotherapeutic agent of the invention. Hypoxic
cytotoxins include, but are not limited to mitomycin C, analogs of
mitomycin C, and drugs of the nitroimidazole class, such as
etanidazole. The chemotherapeutic agents can also be agents whose
cytotoxicity is enhanced under acid pH conditions, particularly
during hyperthermia. Acidotic cytotoxins include, but are not
limited to, cisplatin, analogs of cisplatin, bleomycin, flavone
acetic acid and etanidazole.
According to the present invention, the NO scavenger or NO synthase
inhibitor should be administered so that vascular stasis occurs
after the hypoxic cytotoxin or acidotic cytotoxin has completely
infused within the tumor. In one embodiment, the NO scavenger or NO
synthase inhibitor can be administered simultaneously with
administration of the chemotherapeutic agent. Alternatively, the
agents can be administered sequentially, preferably with the
chemotherapeutic agent administered first, followed by the NO
scavenger or the NO synthase inhibitor. Generally, if administered
sequentially, the NO scavenger or NO synthase inhibitor is
administered about 15 minutes to about 60 minutes after the
chemotherapeutic agent. However, if the NO scavenger or NO synthase
inhibitor is very slow acting, its administration can precede
administration of the cytotoxin.
The present invention also contemplates the administration of an NO
synthase inhibitor, either simultaneously or sequentially with a
hypoxic or acidotic chemotherapeutic agent, followed by the
administration of a NO synthase substrate (e.g., L-arginine), so as
to effect the selective reduction of tumor blood flow and increased
tumor hypoxic cell fraction, while tending to restore normal tissue
blood flow. Preferably, the NO synthase substrate is administered
from 10 minutes to 4 hours after the administration of the NO
synthase inhibitor.
Generally, the dose of the chemotherapeutic agent will be a dose
found to be effective for chemotherapy. For the therapy of a
hypoxic tumor, the dose of the chemotherapeutic agent may be less
than the standard amount administered for chemotherapy.
The dose of the NO scavenger or NO synthase inhibitor to be
administered is a dose effective to reduce blood flow or the level
of oxygenation in a tumor, e.g., as detected by such ability in
vivo, in in vitro assays, or in the animal model systems as
described in Sections 6 and 7, infra. For example, if the NO
scavenger is cell free hemoglobin, the dose (in g of scavenger per
mass of the subject in kg) can be from about 0.01 g/kg to about 10
g/kg; in a specific example, infra, the dose is about 0.1 g/kg,
which represents less that 5% of the total blood volume of the
animal. If the NO synthase inhibitor is a substituted arginine, the
dose can be from about 0.1 mg/kg to about 100 mg/kg. In a specific
embodiment, infra, the synthase inhibitor is L-NMA administered at
a dose of 3.0 mg/kg. In an embodiment in which an NO synthase
substrate is administered, the dose of the NO synthase substrate is
preferably from 30 mg/kg to 300 mg/kg.
In a specific aspect of the invention, the NO scavenger, preferably
cell free hemoglobin, or the NO synthase inhibitor, and a hypoxic
or acidic cytotoxin can be prepared in a pharmaceutical composition
with a pharmaceutically acceptable carrier.
5.3.3. ADMINISTRATION WITH HYPERTHERMIA
In another aspect, the present invention contemplates
administration of an NO scavenger or an NO synthase inhibitor in
conjunction with hyperthermia therapy for the treatment of a solid
tumor. Hyperthermia therapy refers to use of physical agents, such
as but not limited to microwaves, ultrasound, or other heating
element for local or regional heating, or radiant heat for total
body hyperthermia. Generally, tumor vasculature cannot respond to
heat stress as well as normal tissue, and reducing tumor blood flow
enhances this effect. Administration of an NO scavenger or an NO
synthase inhibitor further reduces the ability of tumors to
responds to heat stress. Furthermore, the present methods can help
overcome some of the limitations of hyperthermia therapy that
result from the non-uniformity of the temperature within the tumor,
particularly regions of the tumor with relatively high blood
flow.
Moreover, by reducing tumor blood flow, metabolism in the tumor
becomes more anaerobic, resulting in production of lactic acid and
a decrease in pH. Decreasing pH in a tumor significantly increases
the effectiveness of hyperthermia therapy, e.g., by as much as five
orders of magnitude.
According to the present invention, inhibition of NO activity
precedes hyperthermia therapy so that tumor hypoxia and acidosis of
the tumor can occur or increase by the time of hyperthermia
application. Thus, in a specific aspect of the invention, the NO
scavenger or the NO synthase inhibitor is administered about 10 min
to 12 hours prior to hyperthermia therapy; preferably about 30 min
to about 3 hours prior to therapy; most preferably about 1 hour
before hyperthermia therapy. The dose of NO scavenger or NO
synthase inhibitor to be administered are generally about the same
as the dose administered to enhance hypoxic or acidic cytotoxin
chemotherapy.
In a preferred aspect of the invention, an NO scavenger or NO
synthase inhibitor is administered in conjunction with hyperthermia
therapy and hypoxic or acidic cytotoxin chemotherapy. The NO
scavenger or NO synthase inhibitor is preferably administered so
that blood flow reduction and vascular stasis occurs after infusion
of the cytotoxin in the tumor, and before application of
hyperthermia therapy so that hypoxia and acidosis of the tumor can
occur or increase. It is believed that such an approach is
particularly effective, and provides an increased chance of a
favorable therapeutic outcome.
When an NO synthase inhibitor is used, it is optionally followed by
the administration of an NO synthase substrate, so as to promote
the restoration of normal blood flow in normal tissue.
5.3.4. SOLID TUMORS
The present invention is directed to the use of an inhibitor of NO
activity, such as a nitric oxide scavenger or an NO synthase
inhibitor, as an antitumor therapy to reduce tumor blood flow and
oxygenation or as an adjunct therapy to enhance the effectiveness
of hypoxic or acidic cytotoxins, and of hyperthermia, against
tumors, particularly hypoxic or acidic tumors. The present
invention further contemplates effecting therapeutic treatment of
aerobic tumors, which are normally resistant to hypoxic or acidic
cytotoxins, by decreasing blood flow in such tumors and thereby
increasing the sensitivity of such tumors to hypoxic or acidic
cytotoxins. In a specific embodiment, the invention further
contemplates achieving selective blood flow reduction in tumor
tissue by administering an NO synthase inhibitor, followed by
administration of an NO synthase substrate.
Examples of solid tumors that can be treated according to the
invention include sarcomas and carcinomas such as, but not limited
to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma.
In another embodiment, dysproliferative changes (such as
metaplasias and dysplasias) are treated or prevented in epithelial
tissues such as those in the cervix, esophagus, and lung. Thus, the
present invention provides for treatment of conditions known or
suspected of preceding progression to neoplasia or cancer, in
particular, where non-neoplastic cell growth consisting of
hyperplasia, metaplasia, or most particularly, dysplasia has
occurred (for review of such abnormal growth conditions, see
Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders
Co., Philadelphia, pp. 68-79). Hyperplasia is a form of controlled
cell proliferation involving an increase in cell number in a tissue
or organ, without significant alteration in structure or function.
As but one example, endometrial hyperplasia often precedes
endometrial cancer. Metaplasia is a form of controlled cell growth
in which one type of adult or fully differentiated cell substitutes
for another type of adult cell. Metaplasia can occur in epithelial
or connective tissue cells. Atypical metaplasia involves a somewhat
disorderly metaplastic epithelium. Dysplasia is frequently a
forerunner of cancer, and is found mainly in the epithelia; it is
the most disorderly form of non-neoplastic cell growth, involving a
loss in individual cell uniformity and in the architectural
orientation of cells. Dysplastic cells often have abnormally large,
deeply stained nuclei, and exhibit pleomorphism. Dysplasia
characteristically occurs where there exists chronic irritation or
inflammation, and is often found in the cervix, respiratory
passages, oral cavity, and gall bladder. For a review of such
disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B.
Lippincott Co., Philadelphia.
The present invention is also directed to treatment of
non-malignant tumors and other disorders involving inappropriate
tissue vascularization by administering a therapeutically effective
amount of an inhibitor of NO activity, such as an NO scavenger or
an NO synthase inhibitor, as an adjunct (additional therapy) to
treatment of a disorder that involves inappropriate tissue
vascularization. Inappropriate tissue vascularization includes an
increase in the number of blood vessels or hypertrophy, which is an
increase in the size of the blood vessels. A therapeutically
effective amount of an NO scavenger or an NO synthase inhibitor is
an amount effective to induce resolution of symptoms. In another
aspect, a therapeutically effective amount is an amount effective
to decrease blood flow through the tissue.
For example, it is contemplated that the invention is useful for
the treatment of arteriovenous (AV) malformations, particularly in
intracranial sites. Radiation therapy is commonly used to treat
such inoperable lesions. Use of NO scavengers and/or NO synthase
inhibitors could lead to enhanced rate or frequency of sclerosis or
thrombus formation within these lesions and reduction in size of
the malformation as detected by angiography, thereby increasing the
efficacy of the approach. Reducing the time for the therapeutic
effect to occur reduces the time that the patient is at continued
risk for intracranial hemorrhage. In this case, use of conformal
radiation treatment planning leads to preferential therapeutic
effect within the volume of the AV malformation.
Hyperthermia has been used to treat psoriasis, a dermatologic
condition that is characterized by inflammation and vascular
proliferation. Administration of a NO scavenger or NO synthase
inhibitor may be used to increase the efficacy of this therapy, by
enhancing the rate of thrombosis within the effected vessels, and
preferably to induce disappearance of the lesion. Similarly,
hyperthermia is also being used for treatment of benign prostatic
hypertrophy. This condition is also associated with inflammation
and possibly vascular proliferation. Administration of an NO
scavenger or an NO synthase inhibitor can enhance the therapeutic
benefits of this treatment. Hyperthermia has also been used for the
treatment of cutaneous fungal infections. Administration of an NO
scavenger or an NO synthase inhibitor can enhance the therapeutic
benefits of this treatment, and preferably induce disappearance of
the lesions.
Treatment of other hyperproliferative disorders is also
contemplated.
5.3.5. IRREVERSIBLE REDUCTION IN TUMOR PERFUSION
According to one aspect of the invention, NO synthase inhibition
followed by administration of L-arginine or other NO synthase
substrate provides a means of achieving the therapeutic goal of
selective tumor hypoxia. For example, a NO synthase inhibitor (a
competitive inhibitor, e.g., a substrate analog) can be
administered to a subject having a solid tumor, followed by the
administration of an NO synthase substrate, in order to achieve
selective reduction in tumor blood flow and oxygenation.
In a specific embodiment, a NO synthase inhibitor can be
administered concurrent with or following administration of a
hypoxic or acidotic sensitizer or hyperthermia. This strategy is
used to reduce tumor blood flow, increase hypoxic cell fraction,
and improve retention of the drug within the tumor. Normal tissue
blood flow can subsequently be restored (or at least increased
toward normal levels) by the administration of L-arginine or other
NO synthase substrate, thus reducing toxicity to normal tissue,
while not compromising the therapeutic efficacy in the tumor. The
combination of an NO synthase inhibitor followed by L-arginine or
other NO synthase substrate can also be used to increase tumor
retention of other chemotherapeutic agents by reducing washout, as
well as to reduce thermal washout during hyperthermic therapy of
tumors.
The invention can be better understood by referring to the
following examples, which are provided merely by way of
exemplification and is not intended to limit the invention.
6. PHARMACOLOGICAL REDUCTION OF TUMOR PERFUSION: A MECHANISM TO
IMPROVE THERAPEUTIC EFFECTIVENESS OF BIOREDUCTIVE CHEMOTHERAPEUTIC
AGENTS AND HYPERTHERMIA
6.1. METHODS
6.1.1. GENERAL EXPERIMENTAL PROCEDURE
Fischer 344 rats, weighing 150-200 g and bearing dorsal skinfold
window chambers containing 8 to 10 day R3230AC (rat mammary
adenocarcinoma) tumors, were anesthetized with sodium pentobarbital
(40 mg/kg, intraperitoneal (IP)). The femoral artery and vein were
cannulated for measurement of arterial blood pressure and
intravenous (IV) infusion of drugs. The rats were mounted on the
stage of a Zeiss photomicroscope II equipped with both transmitted
light and epifluorescence capability. Selected vessels in the tumor
center, peripheral tumor, and in normal areas of the window
preparation were observed through either a black and white video
camera or a silicon intensified tube camera and videotaped using a
Super VHS recorder. The same vessels were observed both prior to
treatment (baseline) and following treatments, as described
below.
6.1.2. NO SYNTHASE INHIBITION EXPERIMENTS
In experiments investigating the effect of NO synthase inhibition
on tumor blood flow, the dorsal glass window was removed from the
window chamber and Earle's Balanced Salt Solution (BSS), heated to
36.degree. C. and gassed with 95% N.sub.2 /5% CO.sub.2, was
superfused across the window preparation. Following baseline video
recordings of blood flow, N.sup.G -monomethyl-L-arginine (L-NMA,
Calbiochem cat #475886; 50 and 100 .mu.M in Earle's BSS, at
36.degree. C., bubbled with 95% N.sub.2 /5% CO.sub.2) was
superfused at 1.5-2 ml/min across the exposed face of the tumor for
at least 45 min before video recordings of the same vessels were
made.
In a separate set of experiments, the NO synthase inhibitor L-NMA
was administered IV at 3.0 mg/kg (Kilbourne et al., 1990, Proc.
Natl. Acad. Sci. USA 87:3629-3632) and mean arterial pressure,
heart rate, and tumor and normal vessel blood flows were
monitored.
6.1.3. NO SCAVENGING EXPERIMENTS
In experiments investigating the effect of NO scavenging on tumor
blood flow, cell-free hemoglobin (CFHb, bovine origin, Biopure
Formula 1; 0.1 g/kg) was infused IV. This dose represents less than
5% of the total blood volume of a 150 rat. Again, baseline arterial
pressure, heart rates, and video recordings of vessel blood flow
were made prior to treatment with CFHb (baseline) and following
treatment.
6.1.4. NO SCAVENGING--HYPOXIC SENSITIZER EXPERIMENTS
Experiments investigating the effect of NO scavenging combined with
a hypoxic cell cytotoxin on tumor growth were performed. Human
rhabdomyosarcoma xenographs (DU-217P) were implanted in nude mice.
CFHb (Biopure Formula 1, 0.1 g/kg) was administered IV 60 min
following administration of mitomycin C (15.7 mg/m.sup.2) and the
delay in tumor growth was determined. A Wilcoxon rank sum test was
used to compare treatments with controls. A p value.ltoreq.0.05 was
considered significant.
6.2. RESULTS
6.2.1. NO SYNTHASE EXPERIMENTS SUPERFUSION
Administration of L-NMA via a superfusion medium just to the window
chamber surface did not change systemic cardiovascular function, as
indicated by observed changes in mean arteriolar pressure (MAP).
Average baseline MAP was 105 mm Hg, and average MAP during L-NMA
superfusion was 101 mm Hg. Table 1 summarizes the results of 4
superfusion experiments on tumor and normal vessel blood flow:
TABLE 1 ______________________________________ Effect of L-NMA
Superfusion on Tumor and Normal Vessel Blood Flow Blood Flow Vessel
Location Baseline L-NMA Superfusion
______________________________________ Tumor Flow present No Flow
3/7 N = 7 7/7 vessels Greatly Reduced 2/7 Reduced 1/7 Flow not
changed 1/7 Normal Flow present No Flow 1/8 N = 8 8/8 Flow not
changed 7/8 ______________________________________
6.2.2. NO SYNTHASE INHIBITION EXPERIMENTS INTRAVENOUS
ADMINISTRATION
Intravenous administration of L-NMA caused a transient 20 mm Hg
increase in MAP, peaking at 5 min following administration and
returning to preinjection levels by 30 min, with little or no
change in heart rate (FIG. 1). Table 2 summarizes the results of 2
intravenous L-NMA administration experiments on tumor vessel blood
flow:
TABLE 2 ______________________________________ Effect of
Intravenous L-NMA on Tumor Vessel Blood Flow Vessel Location
Baseline Intravenous L-NMA ______________________________________
Tumor Flow present Flow 4/24 N = 24 24/24 Greatly Reduced 5/24
Reduced 4/24 Flow not changed 11/24 Normal None observed
______________________________________
6.3.3. NO SCAVENGING EXPERIMENTS
Intravenous administration of CFHb caused a transient 35 mm Hg
increase in average MAP, accompanied by a baroreflex induced
decrease in heart rate, from 256 beats/min to 243 beats/min (FIG.
2). Although MAP returned to preinjection levels by 60 min
following CFHb injection, heart rate remained decreased, with some
indication of a trend towards preinjection levels. Table 3
summarizes the results of experiments with intravenously
administered CFHb on tumor and normal vessel blood flow.
Significantly, administration of L-arginine (100 mg/kg IV) did not
reverse the CFHb-induced reduction in tumor vessel blood flow.
TABLE 3 ______________________________________ Effect of
Intravenous CFHb Administration on Normal and Tumor Vessel Blood
Flow Vessel Location Baseline CFHb Treatment
______________________________________ Tumor Flow present No Flow
(9/28) N = 28 28/28 Greatly Reduced (7/28) Slightly Reduced (1/28)
Flow not changed (11/28) Normal Flow present Flow reduced 1/4 N = 4
4/4 Flow not changed (3/4)
______________________________________
6.2.4. NO SCAVENGING--HYPOXIC SENSITIZER EXPERIMENTS
The results of experiments designed to show the effects of an NO
scavenger on hypoxic cytotoxin sensitization are shown in Table 4.
Mitomycin C treatment alone resulted in a 6 day delay in tumor
growth compared with the control (no treatment) group (p<0.001).
CFHb treatment alone resulted in no significant delay of tumor
growth compared with the control group (p=0.342). The combination
of CFHb and mitomycin C resulted in a 10 day delay in tumor growth
compared with the control group (p<0.001). The data clearly show
a trend toward increased enhancement in growth delay with the
combination of CFHb and mitomycin C compared with mitomycin C alone
(4.4 days; p=0.09).
TABLE 4 ______________________________________ Effect of CFHb and
Mitomycin C on Growth Delay of DU-217P Human Rhabdosarcoma
Xenographs Mito- Mitomy- mycin Mitomycin cin and and CFHb C vs CFHb
vs CF vs vs Mito- Treatment Control Control Control mycin
______________________________________ Regressions 1/10 0/10 3/10
N/A .DELTA. 6.03 days 1.16 10.36 4.4 days Treatment- days days
Control (days) P value .ltoreq. 0.001 10.342 10.001 10.09
______________________________________
6.3. CONCLUSIONS
In summary, a series of 11 experiments on the effect of NO
inhibition on tumor blood flow have been performed, which included
59 tumor vessels and 12 normal tissue vessels. Modulation of NO
levels with the NO synthase inhibitor L-NMA or the NO scavenger
CFHb resulted in decreased blood flow in 61% of the tumor vessels
studied, compared with decreased blood flow in 17% of the blood
vessels in normal tissues. Complete vascular stasis resulting from
administration of either the NO synthase inhibitor or the NO
scavenger was observed in 27% of all tumor vessels and 8.3% of
normal tissue vessels. Thus, the effects of administration of the
NO synthase inhibitor or the NO scavenger appear to occur
preferentially, though not exclusively, in tumor tissues.
L-NMA or CFHb-induced vascular stasis in tumor vessels could not be
reversed with L-arginine. This result suggests that the effect of
NO reduction may involve more than vasomotor tone in tumor. For
example, platelet adhesion may be playing a role.
The combination of the NO scavenger CFHb with the hypoxic cytotoxin
mitomycin C demonstrates a clear increase in tumor growth delay
compared to mitomycin C alone, although the increase in not
statistically significant at this time using the Wilcoxon rank sum
analysis.
7. EXAMPLE: CHANGES IN TISSUE AND TUMOR OXYGENATION WITH
ADMINSTRATION OF STROMA FREE HEMOGLOBINS
This Example is a report of physiological studies on the effect of
administration of stroma free hemoglobins on normal tissue and
tumor oxygenation. The results are based on three animals per
experimental group.
7.1. MATERIALS AND METHODS
The three experimental groups were (1) p50 of 9.0 mmHg, (2) p50 of
32.0 mm Hg and (3) Albumin. The concentration of all three
solutions at the time of administration was 10 g per 100 ml. The
dose was 1.5 ml/125 gm body weight administered as a slow infusion
over 10 minutes. All animals had a femoral arterial catheter placed
for continuous monitoring of arterial pressure. Heart rates were
also obtained from the pressure tracings. Clark style
microelectrodes were placed in muscle and in two R3230AC tumor
sites for monitoring of tissue oxygenation after administration of
the various solutions. The R3230AC tumor is a rat mammary
adenocarcinoma (see Section 6.1.1., supra). The tumor was
transplanted into the animal's leg.
All the animals were anesthetized with phenobarbital prior to the
experiments. Body temperature was maintained with at 37.degree. C.
with a thermostatically controlled heating blanket.
7.2. RESULTS
The changes in systemic cardiovascular function are noted in FIG.
3. For all three infusion solutions a mild tachycardia was noted,
which was most prominent for albumin. The tachycardia persisted out
to 60 minutes after the initiation of the experiment (FIG. 3).
There was no difference in the systolic-diastolic pressure
difference for any of the infusion solutions over the 60 minute
sampling interval. This could be interpreted as reflecting no
change in stroke volume. There was a mild decrease in mean arterial
pressure from baseline (100 mmHg) to an average reading of
approximately 85 to 90 mm Hg after infusion of the albumin
solution. We have observed this type of reaction previously with
Fluosol and Ringers solutions, and have attributed it to a
baroreceptor reflex. In contrast, the mean arterial pressures for
both hemoglobin solutions increased following administration (FIG.
4). These effects persisted out to 60 minutes after administration.
Thus, the hemoglobin solutions appear to be offsetting the
baroreceptor reflex. This may be due to the nitric oxide scavenging
availability of the hemoglobin solutions, which is absent from the
albumin solution. All three solutions created hemodilution as was
reflected by a drop in hemocrit. The hemocrit appeared to be fairly
stable once the infusion was completed, thus indicating that the
solutions were isotonic nature (FIG. 5).
Changes in tissue and tumor oxygen tension during and after the
infusion of these solutions are noted in FIGS. 6-9. The
administration of albumin did not seem to effect muscle pO.sub.2 at
all (FIG. 6). In contrast to the results observed in muscle, the
administration of albumin appeared to improve tumor oxygenation
shortly after completion of solution administration (FIG. 7). This
result may be simply due to hemodilution affects which may improve
tumor blood flow via an effect on blood rheology. By comparison
both hemoglobin solutions created a drop in tumor oxygenation.
Interestingly, the most prominent drop occurred after
administration of the 32 mm Hg hemoglobin (FIGS. 7, 8, and 9).
Hemoglobin did not significantly affect muscle oxygenation.
7.3. DISCUSSION
The drop in tumor oxygenation that results from administration of
hemoglobin has clear therapeutic implications in strategies to
selectively kill hypoxic cells. Hemoglobin did not induce a
significant drop in muscle oxygenation, however, thus
administration of stroma free hemoglobin selectively affects tumor
tissue oxygenation, but not normal tissue. The selective nature of
the hemoglobin induced decrease in oxygenation enhances its value
as an adjuvant in hypoxic tumor therapy.
Stroma free hemoglobin scavenges nitric oxide, as shown by its
ability to offset the baroreceptor reflex. NO scavenging is
believed to cause the observed drop in tumor oxygenation as
well.
8. SELECTIVE REDUCTION OF TUMOR PERFUSION WITH NITRIC OXIDE
SYNTHASE INHIBITION
As described herein, we have found that pharmacological inhibition
of vascular NO followed by L-arginine results in selective and
irreversible tumor flow reduction and that normal tissue flow, but
not tumor flow, can be subsequently restored by the administration
of L-arginine. The microvascular effects of 60 min superfusion of
the nonspecific NO synthase inhibitor, N.sup.G monomethyl
L-arginine (L-NMA, 50 .mu.M), followed by 60 min superfusion of the
true substrate for NO production, L-arginine (200 .mu.M), were
determined using video microscopy in female Fisher 344 rats
implanted with dorsal skin flap window chambers and R3230Ac mammary
adenocarcinomas. L-NMA decreased tumor preparation blood flow 43%
and control preparation blood flow 83% through reduction of venule
diameter and red blood cell (RBC) velocity, decreased microvascular
length density, and increased the percentage of tumor vessels
showing intermittent vascular flow and stasis. Although these
changes were reversible with L-arginine in control venules, tumor
venule diameter, RBC velocity, and vessel length density were not
restored.
8.1. MATERIALS AND METHODS
8.1.1. ANIMAL MODEL
Female Fischer 344 rats (Charles River Laboratories, Raleigh N.C.),
weighing 150-200 g, were surgically implanted with cutaneous window
chambers in order to visualize granulating subcutaneous tissue
microvasculature and to provide a substrate for tumor growth.
Details of chamber design and surgical technique have been
published elsewhere (Pappenfus et al., 1979, Microvasc. Res.
18:311-318). Briefly, aseptic surgical dissection of a 1.0 cm
diameter hole was made through opposing surfaces of the dorsal skin
flap, leaving a single fascial plane with two or three artery-vein
pairs. In tumor-bearing preparations, a 0.1 mm.sup.3 piece of tumor
(R3230Ac mammary adenocarcinoma (Hilf et al., 1965, Cancer Res.
25:286-299) was placed onto the fascial plane at the time of
surgery, whereas in control chambers, no tumor was implanted.
Following surgical implantation, animals were housed individually
in an environmental chamber maintained at 34.degree. C. and 50%
humidity with continuous access to food and water. All preparations
were used 9-11 days following surgery, at which time the tumors
were 3-4 mm in diameter.
8.1.2. MEASUREMENT OF VENULE INTRALUMINAL DIAMETER AND RBC
VELOCITY
Measurements of venule diameter and RBC velocity were performed
using video microscopy of the window chamber preparation as it was
transilluminated with a 40 W tungsten source at 200X on a Zeiss
photomicroscope microscope stage (Carl Zeiss, Photomicroscope III,
New York, N.Y.) equipped with a two axis linear measuring system
(2-LM.5, Boeckeler Instruments, Tucson Ariz.). Images were captured
with a video camera (MTI CCD-72, Dage-MTI, Michigan City Mich.) and
recorded on S-VHS tape for later analysis of vessel diameter
(SVO-9500MD, Sony Corporation of America, San Jose Calif.).
Identities and locations of individual vessels and exact location
of RBC velocity measurement were noted by tracing the vascular bed
for each region of interest onto acetate sheets placed over the
video monitor and by noting the x-y position of the field. Vessel
diameter was measured at sites of RBC velocity measurement by using
a frame grabber (PC Vision+, Imaging Technology Inc., Woburn Mass.)
and image analysis software (Java, Jandel Scientific, Conte Madera,
Calif.). The dual window technique was used to measure RBC velocity
(IPM Model 204 Video Analyzer and 102B Velocity Tracker, San Diego
Calif.) (Tompkins et al., 1974, Rev. Sci. Instrum. 45:647-649).
Superimposition of a videotimer signal (CTG-55 Video Timer, For.A
Co., Ltd., Los Angeles, Calif.) was used to document time of the
videotape record relative to treatment. Relative flow was derived
to illustrate the interaction between cross sectional area and
velocity, where: Relative Flow=(fractional change in
diameter).sup.2 * (fractional change in RBC velocity)
8.1.3. DETERMINATION OF VESSEL LENGTH DENSITY AND INTERMITTENT FLOW
RATIO
Videotaped segments of each experiment prior to treatment
(baseline), following treatment with L-NMA, and again following
treatment with L-arginine were used to obtain the morphometric
index vessel length-density (Chen et al., 1981, Am. J. Physiol.
241:H306-H310), and frequency of intermittent vascular flow and
stasis. For determination of vessel length density, a square grid
was superimposed over the video screen and the number of
intersections between the grid and all vessels with RBC flow during
a 1 min period were counted. The number of grid squares per video
field was 408. Typically the number of intersections ranged from
150 to 300 per video field. The vessel length-density in
mm/mm.sup.3 tissue was calculated using:
where N.sub.intersections =number of intersections between vessels
and gridlines, g=number of blocks in grid (408), d=length of one
grid square side corrected for magnification (0.0193 mm), and
t=measured depth of field through which microvessels could be
discerned (0.15 mm). The change in vessel length density with
treatment was determined by dividing treatment length density by
baseline length density.
Percent of venules demonstrating intermittent flow or stasis, where
intermittent flow was defined as stopped or reversed flow for
.gtoreq.5 sec, was also determined from the videotaped experiments.
First, the total number of vessels per field was counted. A vessel
was defined as a segment between branch points. Vessels showing
intermittent flow or stasis were counted. Vessels which could not
be positively identified as having flow were noted separately. The
percent of vessels demonstrating intermittent flow or stasis was
calculated as the number of vessels with intermittent flow and flow
stasis observed at any time during the 1 min observation interval
divided by the total number of vessels in the field minus the
number of vessels with undetermined flow status. Each vessel with
intermittent flow was counted only once, even if it stopped more
than once during the 1 min interval. The percent of venules
demonstrating intermittent flow or stasis was determined for both
L-NMA and L-arginine treatment.
8.1.4. EXPERIMENTAL PROTOCOL
The animals were anesthetized with sodium pentobarbital (40 mg/kg,
i.p.) and kept on a thermostatically controlled blanket at a rectal
temperature of 37.degree. C. (Model 50-7503 Homeothermic Blanket,
Harvard Bioscience, S. Natick, Mass.). The femoral artery and vein
were cannulated for measurement of arterial blood pressure and i.v.
infusion of drugs. Arterial pressure waveforms (Gould P23XL, Gould
Instrument Systems, Cleveland Ohio) and RBC velocity were each
digitized at 200 Hz and recorded to disk for later analysis, with
heart rate and mean arterial pressure determined from the pulsatile
arterial waveform (AT-Codas, Data Instruments, Akron Ohio). Rats
were placed in lateral recumbency on the microscope stage and the
upper window was removed. EBSS (Gibco cat #450-1100EB, Life
Technologies Inc., Grand Island, N.Y.) bubbled with 95% N.sub.2 /5%
CO.sub.2, was superfused across the surface at 1-2 ml/min.
Temperature of the medium at the tissue surface was 32.degree.
C.
Selected fields of venules were observed in the tumor center, the
hypervascular tumor periphery, and in surrounding "normal" areas of
granulating or healing subcutaneous tissue away from the tumor.
Postcapillary venules in granulating tissue were examined in
nontumor-bearing control window chambers, as well. The same vessels
were observed prior to treatment and following treatment. Following
pretreatment observations, 50 .mu.M L-NMA (Calbiochem cat #475886;
in EBSS) was superfused across the exposed face of the tumor for 60
min and measurements were repeated. L-arginine, (Calbiochem cat
#1820; in EBSS), 200 .mu.M, was then superfused across the chamber
for an additional 60 min before measurements of the same vessels
were again made.
8.1.5. STATISTICAL ANALYSIS
Relative changes from baseline diameter, RBC velocity, mean
arterial pressure, and heart rate for control and tumor-bearing
preparations were assessed using a mixed-effects linear model
(Crowder, M. J., Hand, D. J. Analysis of Repeated Measures, London:
Chapman and Hall, 1990). This model accounts for multiple
measurements on each animal by utilizing within-animal and
between-animal sources of variation in the analysis. Means and
standard errors (and 95% confidence intervals) were estimated from
the models, which were fit using the SAS/STAT procedure PROC MIXED
(SAS Institute, Inc. SAS Technical Report P-229, SAS/STAT Software:
Changes and Enhancements, Release 6.07. Cary NC: SAS Institute,
Inc., 1992). All statistical tests of significance were based on
2-sided tests and a significance level of 0.05.
8.2. RESULTS
8.2.1. BASELINE PARAMETERS
Pretreatment diameter and RBC velocity (mean and standard error
[SEM]) for control (n=3) and tumor-bearing (n=15) window chamber
experiments are summarized in Table 5. There were no pairwise
differences in mean diameter or mean RBC velocity between the three
categories of tumor vessels, adjusted for multiple comparisons.
Pretreatment diameter and RBC velocity for normal venules in tumor
chambers were similar to those of control chamber venules.
Superfusion of L-NMA and L-arginine had no effect on relative
change in mean arterial blood pressure and heart rate (Table
6).
TABLE 5 ______________________________________ Baseline
Postcapillary Venular Blood Flow Parameters Mean Mean Number Number
Diameter Velocity Venule of of (SEM (SEM Type Chamber Animals
Vessels .mu.m) mm/sec) ______________________________________
Central Tumor 8 39 32.2 0.55 Tumor (13.2) (0.19) Peripher- Tumor 7
39 22.0 0.56 al Tumor (4.2) (0.28) Normal, Tumor 6 36 34.7 0.32
near (4.6) (0.09) Tumor Normal, Control 3 37 25.5 0.35 no Tumor
(18.5) (0.37) ______________________________________
There were no significant pairwise differences (where p<0.05,
adjusted for multiple comparisons) in mean diameter or mean
velocity between vessels. Tumor-bearing chamber normal vessels were
not significantly different from control chamber normal
vessels.
TABLE 6 ______________________________________ Relative Changes in
Mean Arterial Pressure and Heart Rate After Superfusion of L-NMA
and L-arainine ______________________________________ L-NMA
L-arginine Mean Arterial Mean Arterial Blood Blood Pressure
Pressure Change (95% Change (95% Vessel Chamber CI) CI)
______________________________________ Central Tumor Tumor 0.95
0.93 (0.90-1.00) (0.81-1.06) Peripheral Tumor 0.98 0.97 Tumor
(0.94-1.03) (0.85-1.09) Normal, near Tumor 0.96 0.89 Tumor
(0.91-1.00) (0.77-1.02) Normal, no Control 0.97 0.97 Tumor
(0.88-1.07) (0.75-1.19) ______________________________________
L-arginine L-NMA Heart Rate Heart Rate Change (95% Vessel Chamber
Change (95% CI) CI) ______________________________________ Central
Tumor 0.89 (0.83-0.95) 0.93 Tumor (0.88-0.99) Peripheral Tumor 0.97
(0.85-0.98) 0.96 Tumor (0.90-1.02) Normal, Tumor 0.96 (0.89-1.02)
0.89 near Tumor (0.84-0.95) Normal, no Control 0.90 (0.78-1.03)
0.92 Tumor (0.83-1.02) ______________________________________
There were no significant differences in mean arterial pressure or
heart rate between treatment, vessel type, or tumor and control
preparations.
8.2.2. VESSEL DIAMETER
Superfusion of L-NMA significantly reduced baseline diameter for
all types of tumor preparation venules as well as venules in
non-tumor control preparations (FIG. 10; all p<0.05). There were
no significant pairwise differences between individual vessel types
with respect to relative diameter changes following L-NMA. However,
there was a greater reduction in vessel diameter in non-tumor than
tumor bearing preparations (p=0.02). Superfusion of L-arginine had
a negligible effect in restoring venule diameters toward baseline
in tumor preparations. Venules in control preparations, however,
returned to pretreatment diameter following L-arginine. There were
no significant pairwise differences between tumor preparation
vessels with respect to relative change in diameter following
L-arginine. There was, however, a significant difference in venule
diameter between tumor and control window preparations following
L-arginine (p=0.01).
8.2.3. RBC VELOCITY
Both normal control venules and central tumor venules showed
significant reductions from pretreatment RBC velocity following
L-NMA (FIG. 11; both p<0.05). Compared with normal venules near
the tumor, RBC velocities for tumor center venules and peripheral
tumor venules were both significantly reduced following L-NMA
(p<0.001, p=0.005, respectively). L-NMA caused a significantly
greater reduction in RBC velocity in control preparations as
compared with tumor-bearing preparations (p=0.004). L-arginine
returned RBC velocity toward pretreatment levels in control
preparations, and restored RBC velocity in both peripheral tumor
venules and normal venules near the tumor. RBC velocity in central
tumor venules, however, was further reduced from baseline following
L-arginine (p<0.05). Central tumor venule RBC velocity remained
significantly lower following L-arginine than that of both
peripheral tumor venules and normal venules near the tumor (p=0.05,
and p=0.03, respectively).
8.2.4. VENULE FLOW
Superfusion with L-NMA reduced flow by 43% in both central tumor
and peripheral tumor venules, and reduced flow by 17% in normal
venules near tumors, while non-tumor control venule flow was
reduced by 83% (FIG. 12). L-arginine increased non-tumor control
venule flow, peripheral tumor venule flow, and flow in normal
venules near tumors to nearly 65% of their pretreatment values.
Flow in central tumor venules, however, decreased to 48% of
baseline in the presence of L-arginine. To avoid multiplication of
error inherent in the determination of both diameter and RBC
velocity, statistical analysis was not performed on relative flow
data.
8.2.5. VESSEL LENGTH DENSITY
Although not statistically significant, tumor preparations showed a
tendency toward reduction in vessel length density relative to
baseline with L-NMA treatment (FIG. 13; where p=0.07 for central
tumor venules, and p=0.08 for both peripheral tumor venules and
normal venules near tumors). Vessel length density further
decreased relative to baseline following L-arginine for both tumor
center and peripheral tumor vessels (p=0.01 and p=0.05,
respectively).
8.2.6. INTERMITTENT VASCULAR FLOW AND STASIS
Superfusion with L-NMA increased intermittent vascular flow and
stasis in central tumor vessels relative to baseline (FIG. 14;
p=0.03). Intermittent vascular flow and stasis showed a tendency
toward an increase in peripheral tumor venules and in normal
venules near tumors, as well (p=0.06 for both comparisons).
Treatment with L-arginine returned intermittent vascular flow and
stasis toward pretreatment levels for all vessel types.
8.3. CONCLUSIONS
We have shown that NO synthase inhibition results in selective and
irreversible tumor flow reduction and that normal tissue flow, but
not tumor flow, can be subsequently restored by L-arginine.
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and the accompanying figures. Such modifications are intended to
fall within the scope of the appended claims.
Various publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.
* * * * *